The aim of the present study was to measure the kinetic parameters of skeletal muscle protein synthesis in rats by deuterated water (2H2O). Twenty Sprague-Dawley (SD) rats were labeled by 2H2O through intraperitoneal injection and drinking. At the each end of the 1st, 3rd, 5th, 6th and 10th week after the first 2H2O labeling, four rats were sacrificed by cardiac puncture for blood plasma and quadriceps femoris sampling. Skeletal muscle protein and free amino acids in plasma were purified, hydrolyzed by hydrochloric acid and derived. The deuterium enrichments of 2H-labeled alanyl in skeletal muscle protein and plasma protein-bound 2H-labeled alanine were determined by gas chromatography-mass spectrometry (GC-MS). The fractional synthesis rate of skeletal muscle protein and synthetic dynamic equation were calculated. The fractional synthetic rate of skeletal muscle protein was 12.8%/week, and synthetic dynamic equation was ft = 0.158 × (1 - e-0.128t). The results suggest that the kinetic parameters of skeletal muscle protein synthesis can be measured by 2H2O labeling, and the method can be applied in long-term labeling experiment.